1. Field of the Invention
The present invention relates to maintaining the activity of enzymes, and more specifically to a combination of protein chaperones from a hyperthermopilic Archaeon for extending the durability and activity of an enzyme.
2. Description of the Related Art
Hyperthermophiles are defined as microorganisms that grow optimally at or above 80° C. Their high temperature resistance raises questions regarding the protein chaperones that can fold proteins at very high temperatures. In common with other hyperthermophiles, Pyrococcus furiosus, an archaeon that grows optimally at 100° C., encodes a reduced set of protein chaperones compared with eukaryotes or Archaea with lower growth temperatures (Laksanalamai et al. 2004). In the P. furoisus genome (Robb et al. 2001), two chaperones, the small heat shock protein (sHsp) and the sHsp60 (chaperonin), have been annotated, expressed and characterized. In addition, several putative chaperones, such as prefoldin, HtpX and Nascent peptide Associated Complex (NAC) have been identified (Laksanalamai et al. 2004). The most extensively studied chaperone in P. furiosus is the sHsp, which is an alpha-crystallin homolog with conserved sequence motifs in common with sHsps and crystallins from all domains of life (Chang et al. 1996; Haley et al. 2000; Kim et al. 1998; Laksanalamai et al. 2003; Laksanalamai et al. 2001; van Montfort et al. 2001). Several lines of evidence indicate that sHsps can prevent denatured proteins from aggregating but are unable to refold non-native proteins in a catalytic fashion (Chang et al. 1996; Laksanalamai et al. 2001). Hsp60s on the other hand catalyze ATP-dependent protein folding (Hartl 1996; Hartl and Hayer-Hartl 2002).
The heat shock proteins of the invention, Pyrococcus furiosus (sHSP), confer thermotolerance on cellular cultures and on proteins in cellular extracts during prolonged incubation at elevated temperature, demonstrating the ability to protect cellular proteins and maintain cellular viability under heat stress conditions. Such heat shock proteins are effective to combat enzymatic aggregation and intracellular precipitation during heat stress, and thereby enable enhancement of the utility and stability of enzymes in various applications, such as use of Taq polymerase in polymerase chain reaction (PCR) applications, digestive enzymes in microbial degradative applications, etc.
The PCR is a powerful method for the rapid and exponential amplification of target nucleic acid sequences. PCR has facilitated the development of gene characterization and molecular cloning technologies including the direct sequencing of PCR amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disease disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the exponential amplification of the nucleotide sequence delineated by the flanking amplification primers.
An important modification of the original PCR technique was the substitution of Thermus aquaticus (Taq) DNA polymerase in place of the Klenow fragment of E. coli DNA pol I. The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures which enhance the specificity of primer:template associations. Taq polymerase thus serves to increase the specificity and simplicity of PCR.
However, while the heat shock proteins provides stability of DNA polymerases in high heat conditions, there is a need in the art to provide a composition that has the potential to promote refolding of proteins and assembly for reuse of polymerases, such as the Taq polymerase.